Project Summary Antibiotic resistance is a rapidly emerging global threat and its overuse has led to resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). To address this growing crisis, ?nonantibiotic? approaches using small molecules that interfere with quorum sensing (QS) can be used as promising alternatives. These molecules disrupt QS, which reduce the possibility of developing resistance by attenuating virulence rather than directly killing bacteria. QS is a form of communication that allows bacterial populations to coordinate their gene expression, which leads to pathogenic processes that actively damage host tissue. Pseudomonas aeruginosa is an opportunistic pathogen and is becoming increasingly resistant to current antibiotics. In P. aeruginosa, QS is mediated by N-(3-oxo-dodeceanoyl) homoserine lactone (3OC12-HSL), which binds to its cognate receptor, LasR, a LuxR-type protein that regulates the transcription of over three hundred genes, including those related to bacterial infection. However, despite excellent work in the discovery of novel small molecules to inhibit species- or strain-specific QS in Gram-negative bacteria, there are a limited number of studies on broad-spectrum QS inhibitors. Most of these compounds are narrow target-concentric, possess poor pharmacokinetic properties (PK), and thus are much less likely to be further developed beyond the preclinical space by pharmaceutical companies. The objective of this proposal is to investigate the therapeutic potential of inhibitors of LasR in P. aeruginosa QS as a proof-of-principle model, to ultimately design broad- spectrum, small molecule prophylactic therapies specifically targeted at conserved Trp60 in LuxR-type proteins that would disrupt QS and attenuate bacterial infection. Biologically relevant small molecule analogues of 3OC12-HSL will be identified through in silico modeling studies with an X-ray crystal structure of LasR and will be prepared by chemical synthesis (Specific Aim 1). The design and preparation of candidate inhibitors will not only allow us to interrogate the mechanism by which interactions govern binding in LasR, but also provide a platform to examine virulent phenotypes in vitro and in vivo (Specific Aim 2). Lastly, we propose to examine the effect on global transcriptomics and proteomics, controlled by global regulator LasR, from wild-type and mutant P. aeruginosa in the presence of LasR antagonists (Specific Aim 3). Completion of these aims will lay the groundwork for a novel class of broad-spectrum ?nonantibiotic? drugs to replace traditional antibiotics.